一種有效區(qū)分移植細(xì)胞和宿主細(xì)胞腦損傷模型的建立

楊上川, 董錦潤(rùn), 瞿家桂,3, 胡新天,, 王正波,

(1. 中國(guó)科學(xué)院生物物理研究所 腦與認(rèn)知國(guó)家重點(diǎn)實(shí)驗(yàn)室, 北京 100101; 2 .中國(guó)科學(xué)院昆明動(dòng)物研究所，云南 昆明 650223; 3. 中國(guó)科學(xué)技術(shù)大學(xué) 生命科學(xué)學(xué)院, 安徽 合肥 230026)

一種有效區(qū)分移植細(xì)胞和宿主細(xì)胞腦損傷模型的建立

楊上川1,2, 董錦潤(rùn)1,2, 瞿家桂1,2,3, 胡新天1,2,*, 王正波1,2,*

(1.中國(guó)科學(xué)院生物物理研究所 腦與認(rèn)知國(guó)家重點(diǎn)實(shí)驗(yàn)室,北京100101; 2 .中國(guó)科學(xué)院昆明動(dòng)物研究所，云南 昆明650223; 3.中國(guó)科學(xué)技術(shù)大學(xué) 生命科學(xué)學(xué)院,安徽 合肥230026)

為了區(qū)分移植神經(jīng)細(xì)胞和宿主細(xì)胞, 便于將來在宿主體內(nèi)對(duì)移植細(xì)胞進(jìn)行在體的電生理記錄以及其它方面的研究, 通過機(jī)械損毀的方法, 建立了一種特殊的腦損傷模型。結(jié)果發(fā)現(xiàn), 通過機(jī)械損毀的方法, 在大鼠大腦皮層形成形態(tài)規(guī)則的損傷空洞, 其模型穩(wěn)定, 重復(fù)性好; 在空洞內(nèi)進(jìn)行干細(xì)胞移植, 能夠長(zhǎng)時(shí)間存活, 移植神經(jīng)干細(xì)胞絕大部分細(xì)胞分化為神經(jīng)元, 只有少量細(xì)胞分化為膠質(zhì)細(xì)胞, 而且移植細(xì)胞與宿主細(xì)胞分界明顯; 對(duì)移植細(xì)胞進(jìn)行單細(xì)胞電生理記錄, 記錄到神經(jīng)元放電信號(hào)。這些結(jié)果表明, 通過機(jī)械損毀的方法, 在大鼠大腦皮層成功建立了一個(gè)穩(wěn)定、精確定位移植細(xì)胞與宿主細(xì)胞界限的腦損傷模型。

機(jī)械損毀法; 移植細(xì)胞; 宿主細(xì)胞;神經(jīng)干細(xì)胞; 腦損傷

神經(jīng)系統(tǒng)性疾病, 包括變性疾病、腦卒中后遺癥等多為大量神經(jīng)元缺失, 致使中樞神經(jīng)系統(tǒng)損傷后結(jié)構(gòu)和功能難以恢復(fù), 嚴(yán)重影響了患者的生活質(zhì)量。隨著對(duì)神經(jīng)干細(xì)胞研究的深入, 揭示神經(jīng)干細(xì)胞能夠分化成為神經(jīng)元并促進(jìn)腦組織結(jié)構(gòu)和功能的恢復(fù), 越來越多的研究者熱衷于細(xì)胞替代治療神經(jīng)損傷。

當(dāng)前的研究表明, 神經(jīng)干細(xì)胞不僅能在宿主中樞系統(tǒng)中存活、遷移, 并分化成幾乎所有已知類型的神經(jīng)元(Gage, 2000; Temple, 2001; Wennersten et al, 2004; Alvarez-Dolado et al, 2006; Makri et al, 2010), 而且能和宿主的神經(jīng)元建立突觸聯(lián)系(Lundberg et al, 1997; Gaillard et al, 2007)。另外, 神經(jīng)干細(xì)胞在治療某些神經(jīng)疾病方面已取得明顯進(jìn)展, 向中樞損傷疾病模型動(dòng)物直接移植干細(xì)胞可以明顯改善被試動(dòng)物的癥狀(Svendsen et al, 1997; McDonald et al, 1999; Englund et al, 2002b; Riess et al, 2002; Ikeda et al, 2005; Cui et al, 2008; Morita et al, 2008; Ideguchi et al, 2010)。但也有研究者認(rèn)為,這些改善也許來自神經(jīng)系統(tǒng)自身的代償功能, 也許是移入的干細(xì)胞促進(jìn)了殘存神經(jīng)元的功能恢復(fù)(Lu et al, 2003)。移植存活并有突觸形成的神經(jīng)干細(xì)胞并沒有起作用, 因?yàn)槟X內(nèi)大部分的突觸是處在沒有功能的狀態(tài)。解決這一爭(zhēng)議的最有效方法是利用電生理記錄技術(shù)直接檢測(cè)移植神經(jīng)元是否參與了腦內(nèi)信息的傳遞。目前對(duì)移植細(xì)胞進(jìn)行電生理檢測(cè)主要采取腦片離體電生理的方法(van Praag et al, 2002; Benninger et al, 2003; Rüschenschmidt et al, 2005; S?rensen et al, 2005; Ding et al, 2006), 該方法對(duì)于檢測(cè)移植細(xì)胞在宿主體內(nèi)的功能狀態(tài)還有一定差距。當(dāng)前對(duì)移植細(xì)胞進(jìn)行在體電生理記錄最大的難題是如何找到移植進(jìn)去的細(xì)胞。本實(shí)驗(yàn)擬通過機(jī)械損毀的方法, 建立一個(gè)便于區(qū)分宿主細(xì)胞和移植細(xì)胞的腦損毀模型。

1 材料與方法

1.1 腦皮層損毀實(shí)驗(yàn)及電生理記錄裝置

自制外徑18 Gauge的不銹鋼導(dǎo)管和導(dǎo)管持握器; 不銹鋼硬腦膜切刀和硬腦膜切刀支架; 不銹鋼空洞側(cè)壁切刀和空洞底面切刀; 直徑0.8 mm的不銹鋼空洞塞和0.8 mm的不銹鋼細(xì)胞懸液塞, 以及外徑21 Gauge不銹鋼細(xì)胞移植注射器。國(guó)產(chǎn)M1.2不銹鋼平底螺釘; 自凝牙托粉和牙托水; 大鼠腦立體定位儀(深圳瑞沃德); 1.0和1.4 mm鉆頭(德國(guó)),電鉆(韓國(guó))。電極材料25 μm鉑銥絲(美國(guó)A-M systems); 電生理記錄儀型號(hào)NI PXI 1033(美國(guó)National instruments公司)。

1.2 實(shí)驗(yàn)動(dòng)物

選取20只成年SD大鼠, 體重(250±20) g, 自由采食和飲水, 隨機(jī)分為正常對(duì)照組(2只)、假手術(shù)組(6只)和腦損傷組(12只, 選取其中6只進(jìn)行神經(jīng)干細(xì)胞移植實(shí)驗(yàn)), 分籠飼養(yǎng)。

1.3 大腦皮層損毀

實(shí)驗(yàn)大鼠肌注0.2 mL的阿托品10 min后, 腹腔注射(60 mg/kg)戊巴比妥鈉麻醉。用剃毛剪剃掉大鼠頭頂部的毛, 將動(dòng)物頭顱固定在立體定位儀上。經(jīng)碘酊和酒精局部消毒后, 在大鼠頭頂部區(qū)域,用手術(shù)刀由前向后切開約1.5～2 cm的切口, 裸露出頭頂骨, 清除頂骨上附著的肌肉組織。用立體定位儀, 在欲損毀皮層區(qū)域上方的頭骨表面做一標(biāo)記。接著用直徑1.4 mm鉆頭在標(biāo)記處鉆通顱骨, 造成一個(gè)導(dǎo)管埋置孔。再在導(dǎo)管埋置孔四周, 以導(dǎo)管埋置孔為中心, 3～4 mm為半徑處, 鉆4個(gè)1 mm的牙托水泥增強(qiáng)螺釘埋植孔(勿損傷硬腦膜), 用滅菌生理鹽水沖洗鉆好了的骨孔, 清除鉆孔產(chǎn)生的骨頭殘?jiān)?/p>

在螺釘孔內(nèi)植入M1.2不銹鋼螺釘, 深度為頭骨的厚度或剛好觸及到硬腦膜, 螺釘在顱骨上方外露2～3 mm。隨后用硬腦膜切刀切除導(dǎo)管埋置孔內(nèi)下方的硬腦膜, 暴露出大腦皮層。通過導(dǎo)管持握器將導(dǎo)管下端輕壓在大腦皮層軟腦膜上, 在導(dǎo)管四周及增強(qiáng)螺釘上澆上牙托水泥固定。

待牙托水泥固化后, 通過埋植好的導(dǎo)管, 用腦空洞側(cè)壁切刀和空洞底面切刀, 對(duì)大腦皮層進(jìn)行切除(深度約1.5 mm)和清除切下的皮層組織, 在導(dǎo)管內(nèi)插入空洞塞。假性手術(shù)組, 只是插入堵管, 不行皮層切除。

1.4 神經(jīng)干細(xì)胞移植

一周后, 選取6只大腦皮層損毀的SD大鼠進(jìn)行神經(jīng)干細(xì)胞移植實(shí)驗(yàn)。大鼠肌注0.2 mL的阿托品10 min后, 腹腔注射(60 mg/kg)戊巴比妥鈉麻醉并固定在立體定位儀上。挑選玫瑰花環(huán)神經(jīng)干細(xì)胞,將細(xì)胞懸液調(diào)至密度為5×107個(gè)/μL備用。在大鼠進(jìn)入麻醉狀態(tài)后, 取出導(dǎo)管內(nèi)的空洞塞, 用細(xì)胞注射器直接將體積1 μL的細(xì)胞注射到人工制造的空洞內(nèi), 插入細(xì)胞懸液塞, 即完成神經(jīng)干細(xì)胞移植。隨后每天肌注(10 mg/kg)環(huán)孢霉素A。

1.5 移植神經(jīng)干細(xì)胞電生理記錄

實(shí)驗(yàn)大鼠在神經(jīng)干細(xì)胞移植后1個(gè)月, 在戊巴比妥鈉麻醉狀態(tài)下, 取出導(dǎo)管內(nèi)的細(xì)胞懸液塞, 將記錄電極從導(dǎo)管內(nèi)插入移植有神經(jīng)干細(xì)胞的孔洞內(nèi), 進(jìn)行電生理記錄。

1.6 組織形態(tài)學(xué)檢查

正常對(duì)照組、假手術(shù)組和不行移植細(xì)胞腦損傷組大鼠于損毀后一周, 移植組大鼠于移植細(xì)胞后2個(gè)月, 腹腔注射60 mg/kg的戊巴比妥鈉麻醉, 4%的多聚甲醛經(jīng)心臟灌流, 快速取出腦組織, 在4%的多聚甲醛中后固定, 進(jìn)行大體解剖觀察, 然后冰凍切片, H.E染色和免疫組化實(shí)驗(yàn), 在激光共聚焦顯微鏡下觀察并拍照。

2 結(jié) 果

動(dòng)物死亡率：本實(shí)驗(yàn)所采用的建模方法安全可靠, 在所有實(shí)驗(yàn)組包括正常對(duì)照組、假手術(shù)組和腦損傷組均無大鼠死亡。

大體病理變化：六只腦創(chuàng)傷鼠損傷程度一致,模型穩(wěn)定, 可控性好, 在損毀區(qū)域均可見一個(gè)形態(tài)規(guī)則的人造損毀空洞, 直徑約0.8 mm, 深度約為1.5 mm。

H.E染色光鏡檢查損傷組：皮層損傷一周后, 病灶斷面形態(tài)規(guī)則, 損傷灶的周圍出現(xiàn)明顯的神經(jīng)膠質(zhì)細(xì)胞浸潤(rùn)(圖1)。在損傷病灶空洞區(qū)域邊緣, 出現(xiàn)2～3層神經(jīng)膠質(zhì)細(xì)胞, 利于移植細(xì)胞的存活。

圖1 大鼠腦皮層損毀及移植Fig. 1 The injured rat brain and the neural stem cells transplanted into the lesion site

神經(jīng)干細(xì)胞移植：對(duì)大鼠損毀后一周進(jìn)行GFP標(biāo)記神經(jīng)干細(xì)胞移植, 2個(gè)月后, 灌流動(dòng)物, 取腦。此時(shí), 人工損毀的空洞幾乎不可見, 已經(jīng)被組織填滿, 略有凹陷。

冰凍切片后, 在激光共聚焦顯微鏡下觀察發(fā)現(xiàn),在先前損毀的空洞內(nèi), 充滿移植的GFP細(xì)胞。在移植細(xì)胞的淺表部分, 有部分細(xì)胞出現(xiàn)死亡, 在空洞的底部, 部分細(xì)胞發(fā)生遷移, 遷移向宿主動(dòng)物腦部的深層(圖1)。

對(duì)腦切片進(jìn)行免疫熒光染色發(fā)現(xiàn), 移植細(xì)胞少量分化為神經(jīng)膠質(zhì)細(xì)胞(圖2), 大多數(shù)分化形成了神經(jīng)元。同時(shí), 觀察到有些宿主自身的膠質(zhì)細(xì)胞突起生長(zhǎng)到移植的細(xì)胞組織中(圖3)。

圖2 對(duì)大鼠移植神經(jīng)干細(xì)胞進(jìn)行免疫組化染色Fig. 2 The immuno-histological stained rat brain with transplanted neural stem cells

圖3 對(duì)大鼠移植神經(jīng)干細(xì)胞進(jìn)行免疫組化染色Fig. 3 The immuno-histological stained rat brain with transplanted neural stem cells

圖4 在體記錄大鼠移植區(qū)神經(jīng)細(xì)胞放電Fig. 4 In vivo electrophysiological recording on transplanted nerve cells

對(duì)皮層損毀大鼠移植神經(jīng)干細(xì)胞一個(gè)月后進(jìn)行單細(xì)胞電生理記錄, 記錄到較好的單細(xì)胞自發(fā)放電(圖4)。

3 討 論

建立腦損傷模型的方法多種多樣, 實(shí)驗(yàn)?zāi)康牟煌捎玫姆椒ㄒ膊槐M相同。常用的方法有液壓損傷(McIntosh et al, 1989)、重物打擊損傷法(Edward Dixon et al, 1991; Marmarou et al, 1994)、壓縮氣擊法(Lighthall, 1988), 以及冷凍傷腦水腫模型(Murakami et al., 1999)、藥物損傷模型(Kim et al, 2002; Dauer ＆ Przedborski, 2003)和負(fù)壓吸除損傷(Kesslak et al, 1986)等。這些模型能較好地模擬腦損傷后的生理病理變化, 能夠提供較好的病理模型;但是這些造模方法存在重復(fù)性不高, 動(dòng)物損毀程度不一致等缺點(diǎn), 且重度損傷時(shí), 動(dòng)物死亡率相對(duì)較高。本實(shí)驗(yàn)通過機(jī)械切除損毀的方法, 能夠較好地在大鼠大腦皮層人工損毀出一個(gè)形態(tài)規(guī)則的空洞,相對(duì)于先前的造模實(shí)驗(yàn), 動(dòng)物死亡率較低, 穩(wěn)定性和重復(fù)性好。先前動(dòng)物模型證實(shí)移植神經(jīng)干細(xì)胞能夠在宿主體內(nèi)存活、 遷移和分化, 并能與宿主神經(jīng)元形成突觸聯(lián)系, 在一定程度上改善病理癥狀; 但是由于在先前動(dòng)物模型進(jìn)行神經(jīng)干細(xì)胞移植實(shí)驗(yàn)時(shí), 在活體情況下, 很難利用電生理的方法定位到移植細(xì)胞, 因此不能證明移植細(xì)胞是否與宿主細(xì)胞產(chǎn)生了真正意義上的功能性聯(lián)系。本模型可以較好地將宿主細(xì)胞與移植細(xì)胞加以區(qū)分, 有效地解決了上述難題。

當(dāng)前動(dòng)物實(shí)驗(yàn)及臨床應(yīng)用中所使用的干細(xì)胞移植途徑主要包括局部注射移植、經(jīng)腦脊液注射移植、經(jīng)血液循環(huán)注射移植(Chen et al, 2001; Chu et al, 2003; Jin et al, 2005)。很多學(xué)者認(rèn)為, 將細(xì)胞直接移植到損毀腦組織周圍, 可以把干細(xì)胞全部集中到病灶及其周邊發(fā)揮治療作用, 神經(jīng)功能改善迅速、直接(Lindvall & Kokaia, 2004; Magnus ＆ Rao, 2005; Fullwood, 2007; Marutle et al, 2007)。但是也有學(xué)者認(rèn)為, 神經(jīng)干細(xì)胞直接移植到病灶中, 其生存不好,相反若移植的細(xì)胞與病灶保持一定的距離, 則能較好地存活, 其原因可能是病灶區(qū)大量的細(xì)胞死亡、水腫帶形成及各種炎性反應(yīng)產(chǎn)生, 導(dǎo)致微環(huán)境的改變, 不利于移植細(xì)胞存活(Jeong et al, 2003), 并且移植神經(jīng)干細(xì)胞能夠向受損部位發(fā)生遷移, 利于干細(xì)胞存活并發(fā)揮功能(Kelly et al, 2004)。在本實(shí)驗(yàn)中,我們也是采用局部注射移植, 將神經(jīng)干細(xì)胞直接移植到人工損毀的空洞內(nèi), 2月后處死大鼠后發(fā)現(xiàn), 移植細(xì)胞幾乎填滿人工損毀的空洞, 表明細(xì)胞存活情況較好, 并未出現(xiàn)由于病灶區(qū)的炎癥反應(yīng)環(huán)境而影響到移植細(xì)胞的存活。筆者認(rèn)為, 可能是由于在我們動(dòng)物損毀模型中, 損毀病灶直徑為0.8 mm, 組織營(yíng)養(yǎng)因子能夠較好地滲透到移植細(xì)胞中間來, 并且腦組織中免疫反應(yīng)可能相對(duì)較弱的緣故。

先前人們對(duì)移植細(xì)胞是否與宿主細(xì)胞產(chǎn)生功能性突觸, 大多采用離體電生理腦片記錄技術(shù)(Englund et al, 2002a; Benninger et al, 2003; Rüschenschmidt et al, 2005; Uchida et al, 2005; Anderová, 2006; Prajerova et al, 2010), 這樣得到的結(jié)果不能真實(shí)反應(yīng)移植細(xì)胞在活體動(dòng)物中的作用, 只能證明移植細(xì)胞與宿主細(xì)胞之間形成了突觸, 并且能夠進(jìn)行突觸傳遞。本實(shí)驗(yàn)中, 存活細(xì)胞大多數(shù)分化為神經(jīng)元,少量細(xì)胞分化為神經(jīng)膠質(zhì)細(xì)胞, 分化形成的神經(jīng)細(xì)胞突起能夠向宿主腦內(nèi)延伸生長(zhǎng), 在交界面移植細(xì)胞能夠向宿主腦內(nèi)發(fā)生遷移, 大多數(shù)移植細(xì)胞與宿主細(xì)胞界限明顯, 對(duì)其中兩只埋置電極大鼠進(jìn)行單細(xì)胞電生理記錄時(shí), 記錄到較好的細(xì)胞放電, 說明該模型可以用于移植細(xì)胞在體的電生理記錄。

總之, 通過機(jī)械切割損毀的方法, 在大鼠大腦皮層能夠較好地?fù)p毀出一個(gè)人為的空洞模型, 穩(wěn)定性好, 可重復(fù)性高, 感染性低, 并且在損毀原位進(jìn)行神經(jīng)干細(xì)胞移植, 能夠長(zhǎng)期存活, 并分化為神經(jīng)元和膠質(zhì)細(xì)胞。能夠較好地將移植細(xì)胞和宿主細(xì)胞分開, 利于將來對(duì)移植細(xì)胞進(jìn)行電生理方面進(jìn)一步的研究。

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A traumatic brain injury model for distinguishing between transplanted neural cells and host cellsin vivo

YANG Shang-Chuan1,2, DONG Jin-Run1,2, QU Jia-Gui1,2,3, HU Xin-Tian1,2,*, WANG Zheng-Bo1,2,*

(1. State Kay Laboratory of Brain and Cognitive Science, Institute of Biophysics, the Chinese Academy of Sciences, Beijing 100101, China; 2. Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, China; 3. School of Life Science, University of Science and Technology of China, Hefei 230026, China)

To perform electrophysiological recording and other investigations on transplanted neural cellsin vivo, we used mechanical damage to establish a special traumatic brain injury model that could distinguish transplanted cells from host cells. The morphology of the trauma-induced holes in the cortex of the rat brain was regular. The model was stable and repeatable. Neural stem cells were transplanted into the trauma-induced hole, and were able to survive for a long time. Most of the transplanted cells differentiated into neurons, and only a small amount turned into glia cells. There was a clear boundary between the host cells and the transplanted cells. Single cell electrophysiological recording on transplanted neural cells were detectedin vivo. This study established a stable and repeatable traumatic brain injury model, which could be used to conductin vivoelectrophysiological recording research on transplanted neural cells.

Mechanical damaging method; Neural cell; Host cell; Neural stem cells; Traumatic brain injury

Q42; R-331; R742；Q813.2

A

0254-5853-(2011)04-0421-07

10.3724/SP.J.1141.2011.04421

2011-01-06；接受日期：2011-04-14

“973”項(xiàng)目(2007CB947703)

?通訊作者(Corresponding authors)，E-mail: xthu@mail.kiz.ac.cn; wangzb@mail.kiz.ac.cn

楊上川, E-mail: Yangsc@mail.kiz.ac.cn